RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-5180
Malaria parasiteP. yoelii
Genotype
Transgene
 Transgene not Plasmodium: HA-tagged TurboID fused with an IMC signal peptide
Promoter: Gene model: PBANKA_1324300; Gene model (P.falciparum): PF3D7_1460600; Gene product: inner membrane complex sub-compartment protein 3 (ISP3)
3'UTR: Gene model: Not available; Gene product: Not available
Phenotype Asexual bloodstage;
Last modified: 4 July 2022, 14:06
  *RMgm-5180
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Introduction of a transgene
Reference (PubMed-PMID number) Reference 1 (PMID number) : 35775739
MR4 number
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Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. yoelii
Parent strain/lineP. y. yoelii 17XNL
Name parent line/clone Not applicable
Other information parent line
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The mutant parasite was generated by
Name PI/ResearcherQuian P, Yuan J
Name Group/DepartmentState Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Signal Network, School o
Name InstituteXiamen University
CityXiamen
CountryChina
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Name of the mutant parasite
RMgm numberRMgm-5180
Principal namesee below
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationNo
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Phenotype
Asexual blood stageHA-tagged TurboID expressed in blood stages (see below for more details)
Gametocyte/GameteNot tested
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant expresses TurboID. TurboID was fused with the N-terminal 20 residues of ISP1 (inner membrane complex sub-compartment protein 1; PBANKA_1209400), a known IMC resident protein and is under the control of the promoter of ISP3 (PBANKA_1324300). The expression cassette is introduced into the parasite as an episome.

Published in: bioRxiv preprint doi: https://doi.org/10.1101/2022.01.28.478263

Protein (function)
Biotin ligase (TurboID)-based proximity labelling was used to compile the proteome of the schizont Inner Membrane Complex (IMC) of rodent malaria parasite Plasmodium yoelii. 

Enzyme-catalyzed proximity labelling (PL) coupled with mass spectrometry (MS) offers an alternative approach for proteome discovery. BioID is an engineered bacterial biotin ligase adapted for proximity-based biotinylation of proteins in living cells. The proteins covalently labeled with biotin can be isolated by streptavidin biotin affinity purification followed by MS analysis. BioID has been used to identify the protein components of complexes and organelles in different model organisms However, BioID requires parasite exposure to biotin over a long period (18-24 h), which are not ideal or feasible for certain developmental stages with a short life span. In addition, BioID does not work well at temperatures below 37°C, rendering its application to the mosquito stages of Plasmodium unsuitable. Recently, a new biotin ligase TurboID was developed by directed evolution. Compared to BioID, TurboID is faster and can work under a broader range of temperature. In this study, we applied TurboID-proximity labeling and quantitative MS to obtain a proteome of the inner membrane complex (IMC) in the schizonts of rodent malaria parasite P. yoelii. TurboID was fused with the N-terminal 20 residues of ISP1 (inner membrane complex sub-compartment protein 1; PBANKA_1209400), a known IMC resident protein.

Phenotype
Biotin ligase (TurboID)-based proximity labelling was used to compile the proteome of the schizont Inner Membrane Complex (IMC) of rodent malaria parasite Plasmodium yoelii. In total, 300 TurboID-interacting proteins were identified. 19 of the 22 selected candidates were confirmed to localize in the IMC. To assess whether the identified proteins are indeed localized in the IMC, 22 candidates were selected among the 300 Tb-IMC interacting proteins for subcellular localization analysis. 

TurboID for PL of the IMC was applied to identify new IMC proteins in the schizonts. The HA-tagged TurboID was fused with an IMC signal peptide, the N150 terminal 20 residues of ISP1 (Tb-IMC). The HA-tagged TurboID alone (Tb-cyto) served as a control to indicate non-specific biotinylation, permitting specific identification of IMC and IMC-associated proteins. Both ligases (Tb-IMC and Tb-cyto) were driven by the promoter of gene isp3 and episomally expressed in the asexual blood stages . As expected, the Tb-IMC ligase dominantly co-localized with the IMC protein GAP45 in the IMC. The schizonts expressing Tb-IMC, Tb-cyto, or empty vector (EV: construct without ligase gene) were purified and treated 157 with 100 μM biotin at 37°C for 3 h. Both immunoblot and dot blot assays using streptavidin-HRP detected increased biotinylation in cell extracts in the presence of biotin from the Tb-IMC and Tb-cyto schizonts, but not from the EV group. Furthermore, parasites stained with fluorescent conjugated streptavidin (SA-488) and anti-HA antibody exhibited an IMC distribution (surrounding daughter merozoites) of biotinylated proteins, which co-localized with ligase in Tb-IMC schizonts. The biotinylated proteins and the ligase displayed cytosolic distribution in the Tb-cyto schizonts, while scarce signal was detected in the EV schizonts. Therefore, the Tb-IMC enables the PL of IMC in the living schizonts.

Additional information
To test the activity of the TurboID ligase relative to the BioID ligase for Enzyme-catalyzed proximity labelling (PL) of malaria parasites, we fused a hemagglutinin (HA) tag to the N-terminus of each ligase. These ligases were episomally expressed in the asexual blood stages of P. yoelii under the promoter of the isp3 gene, a gene that is highly transcribed in the schizonts. Immunoblot detected comparable BioID and TurboID expressions in the asexual blood stages. Different from an automatous rupture of mature schizonts of the in vitro cultured P. falciparum, the P. yoelii schizonts displayed an arrest in rupture after maturation in the in vitro condition, which permits PL of mature schizonts. The schizonts expressing each ligase were incubated with 100 μM biotin at 37°C for different time (0.25, 1, 3, and 18 h). Immunoblot using streptavidin-HRP detected robust protein biotinylation in the cell extracts of TurboID-parasites as early as 0.25 h after biotin incubation. In contrast, protein biotinylation in the BioID-parasites appeared at a low 127 level at 3 h and reached a high level at 18 h. Next we tested the temperature compatibility of the two ligases for PL in the parasites. BioID- and TurboID- schizonts were incubated for 18 and 3 h respectively, with 100 μM biotin at different temperatures (4, 22, 30, and 37°C). Biotin-incubated parasites stained with fluorescently conjugated streptavidin revealed that both ligases had similar labelling activity at 30 and 37°C. Notably, only TurboID retained its activity at 22°C.



Other mutants
  Transgene: Mutant parasite expressing a transgene
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Type and details of transgene
Is the transgene Plasmodium derived Transgene: not Plasmodium
Transgene nameHA-tagged TurboID fused with an IMC signal peptide
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Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/constructCircular plasmid
PlasmoGEM (Sanger) construct/vector usedNo
Modified PlasmoGEM construct/vector usedNo
Plasmid/construct map
Plasmid/construct sequence
Restriction sites to linearize plasmid
Selectable marker used to select the mutant parasitehdhfr/yfcu
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationCRISPR/Cas9 plasmid pYCm was used for parasite genomic modification.
Additional remarks selection procedure
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Other details transgene
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Promoter
Gene Model of Parasite PBANKA_1324300
Gene Model P. falciparum ortholog PF3D7_1460600
Gene productinner membrane complex sub-compartment protein 3
Gene product: Alternative nameISP3
Primer information details of the primers used for amplification of the promoter sequence  Click to view information
Primer information details of the primers used for amplification of the promoter sequence  Click to hide information
Sequence Primer 1
Additional information primer 1
Sequence Primer 2
Additional information primer 2
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3'-UTR
Gene Model of Parasite Not available
Gene productNot available
Gene product: Alternative name
Primer information details of the primers used for amplification the 3'-UTR sequences  Click to view information
Primer information details of the primers used for amplification the 3'-UTR sequences  Click to hide information
Sequence Primer 1
Additional information primer 1
Sequence Primer 2
Additional information primer 2
Insertion/Replacement locus
Replacement / InsertionNot available
Gene Model of Parasite Not available
Gene productNot available
Gene product: Alternative name
Primer information details of the primers used for amplification of the target sequences  Click to view information
Primer information details of the primers used for amplification of the target sequences  Click to hide information
Sequence Primer 1
Additional information primer 1
Sequence Primer 2
Additional information primer 2
Sequence Primer 3
Additional information primer 3
Sequence Primer 4
Additional information primer 4
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Conceptual design of the database: Dr. C.J. Janse (Leiden Malaria Research Group, Leiden, The Netherlands).
Technical design and realization: S. Mededovic and A. van Wigcheren